Photoluminescent semiconductor materials

ABSTRACT

Semiconductor materials having a porous texture are modified with a recognition element and produce a photoluminescent response on exposure to electromagnetic radiation. The recognition elements, which can be selected from biomolecular, organic and inorganic moieties, interact with a target analyte to produce a modulated photoluminescent response, as compared with that of semiconductor materials modified with a recognition element only.

FIELD OF THE INVENTION

[0001] The present invention relates to materials for photodetection andidentification of target analytes, in particular, to materials havinglight-emitting properties and, more particularly, to photoluminescentsemiconductor materials for photodetection and identification of targetanalytes.

BACKGROUND OF THE INVENTION

[0002] Porous silicon (PSi) has been extensively studied for a number ofsemiconductor applications since it was discovered in the late 1950's.More recently, PSi has been shown to exhibit strong visible luminescence(Canham Appl Phys Lett 57:1046; 1990), suggesting promising applicationsin silicon-based opto-electronic devices. Other porous semiconductormaterials such as gallium arsenide, for example, have also been studiedto a lesser extent (Schmuki et al Appl Phys Lett 72:1039; 1998).

[0003] U.S. Pat. Nos. 5,338,415 and 5,453,624 (Sailor et al) describe amethod for detection of chemicals by reversible quenching of PSiphotoluminescence and a device for detection of organic solvents by PSiphotoluminescence, respectively. A silicon wafer was electrochemicallyetched (anodization) with a 50:50 ethanol/hydrofluoric acid (HF)solution to produce a PSi wafer. When the PSi wafer was illuminated witha laser light source in the presence of an organic compound, such astetrahydrofuran (THF), diethyl ether, methylene chloride (MeCl₂),toluene, o-xylene, ethanol and methanol (MeOH), the inherent luminescentemission intensity of the PSi was significantly decreased (i.e., thephotoluminescent response of the PSi was quenched). Also, Sailorobserved that a toluene based solution of ferrocene (i.e.,dicyclopentadienyl Fe (II)) resulted in a complete loss of luminescence.

[0004] Generally, Sailor suggests that the degree of quenching in thephotoluminescent (PL) response tracks with the dipole moment of thecompounds evaluated. Accordingly, these studies suggest that such anevaluation technique can help assess the differences in dipole momentsbetween certain organic compounds. However, Sailor fails to suggest howsuch a method could distinguish between two or more compounds havingsimilar dipole moments, but otherwise very different chemicalcompositions. For example, in U.S. Pat. No. 5,338,415, Sailor observedthat MeCl₂, MeOH and THF all had luminescent quenching ratios of about0.1 or less. Consequently, there is only a small difference in the PLresponse curves produced by each of these compounds, which could be usedto better characterize their respective chemical structures. Also,Sailor discloses a reversible wavelength (“λ”) shift of about 30nanometers (nm, 10⁻⁹ m), from 670 nm to 630 nm, when PSi is exposed toTHF. He suggests that the other organic compounds evaluated producereversible quenching, but fails to suggest that the λ shift is similarto the λ shift observed for THF. However, on its face, it appears thatSailor is suggesting that the λ shifts are substantially similar inmagnitude and direction for all organic compounds evaluated.Accordingly, this 30 nm range provides a somewhat limited window forspectroscopically discriminating between unknown compounds.

[0005] Lin et al describe a biosensor based on induced wavelength shiftsin the Fabry-Perot fringes in the visible light reflection spectrum of athin flat film of PSi (Science 278:840; 31 October 1997). Optically flatthin films of PSi, prepared by electrochemical etching with a 98%ethanol: 49% aqueous HF solution, are sufficiently transparent todisplay Fabry-Perot fringes in their optical reflection spectrum. Arecognition element is immobilized on the flat PSi film. Subsequentbinding of an analyte to the recognition element therefore results in achange in the refractive index of the PSi film and is detected as acorresponding shift in the interference pattern. The interferencepattern is created by reflectance of white light and an interferencepattern produced when multiple reflections of white light are directedtoward a solution/PSi interface and a PSi/bulk silicon interface.Producing and maintaining nearly perfectly parallel planes between theair/PSi and PSi/bulk silicon interfaces is critical to producing preciseand accurate interferometric spectra. Consequently, this technique islimited to applications where environmental conditions such asvibration, temperature and atmospheric gases can be preciselycontrolled.

[0006] Janshoff et al (J Am Chem Soc 120:12108-12116; 1998) alsodescribe PSi for biosensor applications utilizing a shift in aFabry-Perot fringe pattern, created by multiple reflections ofilluminated white light on the air/PSi layer and PSi/bulk siliconinterface, as a means for detecting molecular interactions of species insolution with immobilized ligands as receptors. Janshoff et al statethat “the prerequisite for using porous silicon as an opticalinterferometric biosensor is to adjust the size as well as thegeometrical shape of the pores by choosing the appropriate etchingparameters” (p. 12108). Thin films of silicon were made porous usingelectrochemical etching to produce pores having radii varying from 3 to10 nm, a uniform depth, and cylindrical shape with an absolute surfacearea of about 0.1 to 0.15 m² for samples etched into a 1 cm² patch ofsilicon. Excessive porosity was found to be unsuitable for thebiosensors of Janshoff et al.

[0007] Because interferometric techniques exploit a physical phenomenon,namely, reflectance of light by two different planes to produce aninterference pattern, biosensor systems relying on shifts in theinterference pattern as a means for detecting the presence of an analyteare typically very sensitive to vibration, temperature and atmosphericpressure changes. Furthermore, the reflective plates of the film orwafer, i.e. the air/PSi and the PSi/bulk silicon interfaces, must beparallel, otherwise an undesired shift in the interference pattern canoccur. Typically, the reflective plates of the PSi film must be parallelto 25 Å (2.5 nm). This demands a high level of perfection in manufactureof the PSi wafer or film. Finally, for optimum performance, theirradiated light directed on the PSi film or wafer should beperpendicular to the reflective plates. Accordingly, pores whichthemselves are not perpendicular to the reflective plates affect theinterference pattern of the PSi film or wafer and therefore adverselyaffect results obtained from such biosensors.

[0008] It would therefore be desirable to have a material useful fordetecting target compounds, which can use the photoluminescenceproperties of porous semiconductor materials. Moreover, such materialcould be modified to provide increased sensitivity to quantitativelydetecting low concentrations of predetermined target compounds.

SUMMARY OF THE INVENTION

[0009] According to one aspect of the present invention, there isprovided a modified semiconductor composition comprising: (a) at leastone a semiconductor material having a porous texture and (b) at leastone recognition element, whereby when said composition is irradiatedwith at least one wavelength of electromagnetic radiation in the rangeof from about 100 nm to about 1000 nm, said composition produces atleast one first luminescent response in the range of from about 200 nmto about 800 nm.

[0010] According to another aspect of the present invention, there isprovided a method for producing a luminescent response, comprising: (a)providing a modified semiconductor composition comprising asemiconductor material having a porous texture and at least onerecognition element; (b) irradiating at least said modifiedsemiconductor composition with at least one wavelength ofelectromagnetic radiation in the range of from about 100 nm to about1000 nm to produce at least one first luminescent response; andmeasuring at least the intensity or wavelength of said at least onefirst luminescent response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In drawings which illustrate embodiments of the presentinvention:

[0012]FIG. 1 is a schematic representation of one embodiment of anapparatus which can be used to detect photoluminescence fromphotoluminescent devices of the present invention;

[0013]FIG. 2 is a scanning electron micrograph of a porous siliconparticle produced in Example 1, at a magnification of 300×;

[0014]FIG. 3 is a scanning electron micrograph of a porous siliconparticle produced in Example 1, at a magnification of 800×;

[0015]FIG. 4 is a scanning electron micrograph of a porous siliconparticle produced in Example 2, at a magnification of 800×;

[0016]FIG. 5 is a scanning electron micrograph of a porous siliconparticle produced in Example 2, at a magnification of 5000×;

[0017]FIG. 6 is an epi-fluorescence micrograph at 200× magnification,showing the interaction of antigen to PSi particles modified with IgGantibody, discussed in Example 10;

[0018]FIG. 7 graphically compares acetylcholine hydrolysis for a controlenzyme versus an enzyme-modified PSi, discussed in Example 13;

[0019]FIG. 8 is an epi-fluorescence micrograph at 200× magnificationwhich shows binding of antibody to PSi not treated with glycine buffer,discussed in Example 16;

[0020]FIG. 9 is an epi-fluorescence micrograph at 200× magnificationwhich demonstrates the reduction in binding of antibody of PSi treatedwith glycine buffer, discussed in Example 16;

[0021]FIG. 10 is an epi-fluorescent micrograph at 200× magnificationwhich shows binding activity of antibody in FIG. 9 is not adverselyaffected by glycine buffer, discussed in Example 16;

[0022]FIG. 11 is a graphical representation of photoluminescenceintensity of porous silicon particles discussed in Example 17;

[0023]FIG. 12 is a graphical representation of photoluminescenceintensity of porous silicon particles having a recognition elementattached thereto discussed in Example 17; and

[0024]FIG. 13 is a graphical representation of photoluminescenceintensity of porous silicon particles having a target analyte attachedto a recognition element discussed in Example 17.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0025] As disclosed herein, the porous semiconductor (PSc) materials ofthe invention have photoluminescent (PL) properties and can be used in avariety of analyte detection applications, including, withoutlimitation, biotechnology, such as biosensors for medical,environmental, industrial and defense applications (e.g. chemical andbiological warfare agent detection/identification), genomics,diagnostics, and other molecular/cell biological areas, such as cellisolation/sorting, microstructural cell analysis, etc. In accordancewith the invention, the PSc materials have at least a PSc substrate,which substrate has a porous texture, modified with at least onerecognition element. The PSc substrate is modified with a recognitionelement for interaction with a target analyte (i.e., the compound ofinterest to be detected). The recognition element typically will, butmay not always, modify the PL response of the PSc substrate by producinga λ shift and/or change in the PL intensity as compared to the PScsubstrate free of the recognition element. A PSc modified with suchrecognition elements (“PSc/RE”) can interact with a target analyte sothat a wavelength shift and/or change in PL intensity relative to the PLresponse for PSc/RE is produced, hereinafter referred to simply as,modulation of the PL response or PL modulation, for brevity.

[0026] PSc Substrate

[0027] Preferably, the PSc substrate is comprised of silicon. However,the PSc substrate may comprise any semiconductor material composition,which has photoluminescence properties when made porous. For example,such semiconductor material compositions may include, withoutlimitation, cadmium, copper oxide, germanium, gallium, gallium arsenide,selenium, silicon, silicon carbide, silicon dioxide, silicon galliumphosphide and combinations thereof. The selected semiconductor materialcompositions may also incorporate a dopant, including, for example,without limitation, erbium, boron, phosphorous, copper, phosphors of thelanthanides series, including ytterbium, holmium and thulium, andcombinations thereof. Also, the selected semiconductor materialcompositions may be processed with another compound, including, forexample, without limitation, a halogen, such as bromine, to modify theemission wavelength (Bressers et al J Electro-analytical Chemistry,406:131;1996). For ease of discussion, it should be understood that“PSc” includes, without limitation, any semiconductor materialcomposition, such as those described above for illustrative purposes.

[0028] PSc Structures

[0029] By “porous” or “porous texture” we mean any perturbation, such asdepressions, protrusions or combinations thereof, in or on thesemiconductor material that contributes to the total surface area of thesemiconductor material. Examples of depressions include, withoutlimitation, pores, pits, cavities, craters, trenches, furrows andcombinations thereof. Examples of protrusions include, withoutlimitation, ridges, bumps, bulges, domes, spikes, mounds, mounts andcombinations thereof. For example, without limitation, suchperturbations can be in the form of an ordered honeycomb pore structurecomprised of cylindrical or polygonal shaped pores or a more random porestructure that is coral-like or sponge-like.

[0030] The shape and geometry of the semiconductor material'sperturbations can be diverse. The depressions can range fromregularly-defined shapes and geometries to irregularly-defined shapesand geometries and combinations thereof. Also, the protrusions can rangefrom regularly-defined shapes and geometries to irregularly-definedshapes and geometries. Regularly-defined shapes include, withoutlimitation, circular, semi-circular, ellipsoidal, semi-ellipsoidal,polygonal, square, rectangular, triangular, rhomboidal, trapezial andtrapezoidal shapes. Irregularly-defined shapes include, withoutlimitation, blends of the aforementioned perturbation shapes. Also, forexample, without limitation, the 3-dimensional (3-D) geometry of theperturbations can range from regularly-defined 3-D geometries toirregularly-defined 3-D geometries. Regularly-defined 3-D geometriesinclude, without limitation, cylindrical, conical, cubical,parallelepipedal, polyhedral, rhombohedral, ellipsoidal, helical,spherical, ovoidal, and pyramidal shapes. Irregularly-defined 3-Dgeometries include, without limitation, blends of the aforementionedshapes. In any case, such perturbations in the semiconductor materialresult in an increased surface area of PSc substrate.

[0031] The overall geometric shape of the PSc substrate may be in theform of a film or wafer or have a 3-D structure, relative to a film orwafer. By “3-D structure” we mean that the geometry of the PScsubstrate, whether alone or in combination with a supporting core of anon-PSc material, can range from a regularly-defined geometric shape toan irregularly-defined shape and combinations thereof. Examples ofregularly-defined shapes include, without limitation, spheres,semi-spheres, ellipsoids, semi-ellipsoids, cylinders, ovoids,semi-ovoids, rods, disks, cones, cubes, parallelepipeds, polyhedrons,rhombohedrons and pyramids. Examples of irregularly-defined shapesinclude, without limitation, blends of the aforementioned PSc substrateshapes. For ease of discussion, reference to “PSc structure(s)” hereinwill mean, without limitation, PSc substrate(s), which are in the formof a film or wafer, or which have at least one of the types of thegeometric shapes and porous texture described above for illustrativepurposes.

[0032] FIGS. 2 to 5 are illustrative of just a few of the surfaceperturbations used to enhance the performance of the PSc substrate inthe invention. It will be apparent to those skilled in the art thatthere can be a wide array of perturbations that can be developed in oron the PSc substrate. As discussed more fully below, regardless of theparticular shape or geometry of such perturbations, it is believed thatthese perturbations are a contributing factor to the PL intensityultimately produced by the PSc material. Further, without being bound bytheory, it is believed that the projections of the perturbations are acontributing factor to the PL intensity.

[0033] In accordance with the preferred embodiments of the invention,the PSc substrate has a porous texture on one or more surfaces of and/orthroughout its structure.

[0034] The actual form and/or size of the PSc structure is dependent onthe particular application in which the PSc structures are used. Thereare some applications in which a planar PSc structure is desirable andothers where a 3-D PSc structure is more desirable. When the PScstructure is a 3-D structure, the average diameter or other largestaverage dimension is preferably in the range of from about 100 nm toabout 1 mm. More preferably, the 3-D PSc structures have an averagediameter or other largest average dimension in the range of from about100 nm to about 500 microns. Most preferably, the 3-D PSc structureshave an average diameter or other largest average dimension in the rangeof from about 1 micron to about 500 microns. Furthermore, when the PScstructure is a 3-D PSc structure, the PSc structures are adaptable to avariety of photodetection device configurations, especially wherereduced size is desired for such a photodetection device. For example,in a capillary flow-through column, the diameter of the 3-D PScstructures would preferably be in the range of from about 5 microns toabout 15 microns.

[0035] The PSc structures of the present invention, when irradiated withat least one wavelength of electromagnetic radiation, in the range offrom about 100 nm to about 1000 nm, of sufficient power, produceluminescent radiation in the range of from about 200 nm to about 800 nm,preferably in the range of from about 350 nm to about 700 nm, and morepreferably in the range of from about 450 nm to about 650 nm.

[0036] The PSc structures of the invention can be formed by a variety ofprocedures that will be apparent to those skilled in the art. Planar orwafer-like semiconductor material is commercially available, for examplefrom Silicon Quest (Santa Clara, Calif., USA). Also, for example,non-porous silicon spheres can be produced by the process described inWO98/25090 (Ishikawa, Jun. 11, 1998). Further, a planar or wafer-likesemiconductor material can be mechanically fragmented to produce fineparticles having irregularly-defined geometries. In any case, regardlessof the procedure selected to produce the semiconductor material, suchmaterial can be made porous by a variety of procedures known to thoseskilled in the art, such as those discussed more fully below, forillustrative purposes.

[0037] After a substantially nonporous semiconductor structure isformed, the semiconductor structure is made porous. The desired poroustexture can be produced by using a number of techniques known to thoseskilled in the art. For example, without limitation, a porous texturecan be produced by epitaxial deposition or lithography (Canham Appl PhysLett57:10:1046-1048; 1990), chemical etching (Sailor et al AdvMater9:10:783-793; 1997), anodic etching in HF solutions (Canham ApplPhys Lett 57:10:1046-1048; 1990), spark-erosion (Kurmaev et al J ofPhysics Condensed Mater 9:2671; 1997), laser ablation (Yamada et alJapanese J Appl Physics Part I-Regular Papers Short Note 35:1361; 1996),ion beam milling (Schmuki et al Phys Rev Lett 80:18:406.04063; 1998),and controlled annealing and etching (Tsai et al Appl Phys Lett 60:170;1992). Also, it will be apparent to those skilled in the art thatprocedures used for developing a porous texture on a substantiallyplanar semiconductor material may require some modifications. Forexample, 3-D semiconductor structures may require suspension in an inertgaseous or liquid environment in order that all surfaces be in contactwith a high energy source, etching solution or other means to producethe desired PSc structure. Also, the time required for etching may alsobe much shorter for 3-D PSc structures.

[0038] Also, the PSc structures can be formed with a core and a PScmaterial applied to the core as a coating. Examples of suitable corematerials include, without limitation, glass, plastic, ceramics,zeolites, metals, a different semiconductor material and combinationsthereof. The core material may be selected, for example, to impart adesired buoyant density for a particular application. Such a core islikely to be more suitable when the structures have a larger diameter.In the case of a single semiconductor coating, preferably such a coatingshould be sufficiently thick to produce the desired porous texture.

[0039] A coating of a PSc material may be supported on a core of non-PScmaterial by a variety of techniques. For example, a substantiallynonporous coating of silicon dioxide or silicon carbide can be producedby controlled oxidation (Fauchet J Luminescence 70, 294; 1996) or byhigh temperature pyrolysis techniques (Liu et al Solid StateCommunications 106:211; 1998). Thereafter, the semiconductor materialmay be converted to a porous state, thereby transforming the coated corematerial to a PSc structure. Alternatively, a PSc structure may beformed concurrently with the coating's application to the surface of thecore material. Also, it may be desirable to have multiple layers ofdiffering PSc compositions on the supporting core material.

[0040] Recognition Element

[0041] In the present invention, the PSc structure is modified with atleast one recognition element, and preferably, multiple recognitionelements. For ease of reference, a PSc structure modified with at leastone recognition element hereinafter will be referred to as “PSc/RE”. By“modified”, we mean that a recognition element communicates with the PScstructure such that a PL response of the PSc/RE is modulated when atarget analyte interacts with the recognition element(s) of the PSc/RE.One example of such modification is covalent bonding of a recognitionelement to the PSc structure. However, a direct or physical bond may notbe required. There may be a proximate association of the recognitionelement with the PSc structure sufficient to support electron or energytransfer between the recognition element and the PSc structure.Typically, but not necessarily always, the PSc/RE will exhibit anenhanced PL response relative to the PL response for the PSc structureprior to modification with the recognition element.

[0042] Generally, recognition elements can be organic, inorganic,biomolecular moieties and combinations thereof.

[0043] Preferably, recognition elements are biomolecular moieties.Examples of biomolecular moieties that may be used as recognitionelements are, without limitation, natural or synthetic proteins, nucleicacids, oligonucleotides, lectins, carbohydrates, glycoproteins andlipids, which interact with a target analyte. Proteins are preferred asrecognition elements. Examples of suitable proteins are, withoutlimitation, immunoglobulins, such as polyclonal and monoclonalantibodies, and enzymes. Preferred proteins as recognition elements areimmunoglobulins and enzymes. Examples of suitable nucleic acids includesingle-stranded DNA and double-stranded DNA. A recognition element mayalso have a redox moiety attached thereto. Examples of redox moietiesinclude, without limitation, transition metals and complexes thereof,and co-enzymes such as NAD(H) or NADP(H). The redox moiety may be eitheran electron donor or acceptor to alter PL intensity on binding of atarget analyte to the recognition element.

[0044] Examples of inorganic moieties that may be used as recognitionelements are, without limitation, doped or un-doped crystallineinorganic compounds, for example, linear allotropes of carbon, includingcarbynes and diamond crystals.

[0045] Examples of organic moieties that may be used as recognitionelements are, without limitation, intrinsically conductive polymers(“ICP”), such as polyaniline, and polymer electrolytes, such aspoly[ethylene oxide], advantageously with a lithium triflate dopant.

[0046] Modification of the PSc structure with a recognition element maybe enhanced by using a surfactant to reduce the surface tension of asolution containing the recognition element. Such agents include,without limitation, alcohols and detergents, at a concentrationsufficient to reduce the surface tension of the solution withoutadversely affecting the recognition element's structure or the efficacyof the recognition element or PSc/RE.

[0047] Target Analyte

[0048] A target analyte interacts with the PSc/RE, such that the PLresponse is modulated. By “interact”, we mean that the target analytecommunicates with the recognition element, such that a PL response ofthe PSc/RE is modulated. Examples of such interactions include, withoutlimitation, covalent bonding, hydrogen bonding, Van der Waal's/dipolebonding and affinity binding. However, a direct or physical bond may notbe required. There may be a proximate association of the target analytewith the PSc/RE sufficient to support electron or energy transferbetween the target analyte and the PSc/RE.

[0049] A target analyte may be an organic, inorganic or biomolecularcompound or moiety. Examples of target analytes include, withoutlimitation, (1) antigenic compounds from which antibodies can beproduced including, for example, without limitation, toxins, metabolicregulators, microorganisms, such as bacteria, viruses, yeast, fungi andmicrobial spores, and animal and plant cells/tissue elements; (2)specific substrates for enzymes including, for example, withoutlimitation, metabolites, nerve agents, pesticides, insecticides; (3)complementary oligonucleotide sequences; (4) single-stranded DNA andRNA; and (5) ligands to hormonal receptors and lectins.

[0050] Upon binding of a target analyte to a specific recognitionelement on a PSc structure, the PL response of the PSc structure ismodulated relative to that of the PSc/RE PL response. Preferably, the PLresponse is enhanced relative to that of the PSc/IRE PL response. It ispossible to determine modulation of the PL response in a real- or nearreal-time manner.

[0051] Stabilization/Activation of PSc Structure

[0052] In order to promote stable and efficient modification of the PScstructure with a recognition element, the surface of the PSc structureis first stabilized against uncontrolled oxidation.

[0053] One such stabilization procedure is oxidation using, for example,without limitation, thermal oxidation (Petrova-Koch et al Appl Phys Lett61:943;1992), chemical oxidation (Nakajima et al Appl Phys Lett61:46;1992, Lee et al Mater Res Soc Symp Proc 338:125; 1995, Anderson etal J Electrochem Soc 140:1393; 1993, Duvault-Herrera et al Colloids Surf50:197; 1990, Yamana et al Electrochem Soc 137:2925;1990, Li et al ApplPhys Lett 62:3192;1993), and ozone oxidation processes (Janshoff et al JAm Chem Soc 120:12108;1998). These processes generate reactive hydroxylgroups. Chemical oxidation can be achieved, for example using peroxide,dimethylsulfoxide (DMSO) or iodine chips.

[0054] Covalent Bonding

[0055] As noted above, in one preferred embodiment of the invention, thePSc structure may be modified with a recognition element by covalentbonding. For example, a recognition element may be covalently bonded toa PSc structure using one or more linkers. Linkers can provide atransition in functionality of reactive groups on the PSc structure toan appropriate functional group for attachment of a recognition element.Also, among other functions, linkers can provide a spacer to reducepotential steric hindrance problems arising from a bulky target analyteseeking to interact with a recognition element and/or PSc/RE.

[0056] Preferably, a recognition element is covalently bonded tolinker(s) so that the recognition element and/or PSc/RE can interactwith the target analyte with maximum efficacy. For example, somebiomolecular moieties, such as certain antibodies, linked through theirsulfhydryl groups will exhibit good binding ability for a targetanalyte. However, when such antibodies are attached to a linker throughtheir amine groups, they may have a reduced capacity for interactionwith a target analyte.

[0057] Primary Linker

[0058] In another preferred embodiment of the invention, a primarylinker is attached to hydroxyl groups arising from oxidation of the PSc.One example of a primary linker is a substituted silane. An example of asuitable substituted silane is, without limitation,glycidoxypropyltrimethoxysilane. This primary linker provides a directlink between the hydroxyl groups of an oxidized PSc structure and anamine group of a recognition element. Other suitable primary linkers arehydrosilylated alkenes and alkynes.

[0059] Other linkers which are reactive with hydroxyl groups of theoxidized PSc and amine groups of the recognition element will beapparent to those skilled in the art. It will be understood that alinker may be selected to react with a functional group, other than anamine group, of a recognition element. Other recognition elementfunctional groups include sulfhydryl, carbohydrate or carboxyl groups.It will also be understood by those skilled in the art that otherlinkers, which are reactive with non-hydroxyl groups (produced by otherPSC stabilization techniques) and the functional group of the selectedrecognition elements, may be selected for bonding to a PSc structure.

[0060] Primary & Secondary Linkers

[0061] In another preferred embodiment, a primary linker is attached tohydroxyl groups arising from oxidation of the PSc and then a secondarylinker is attached to the primary linker. One example of a primarylinker that may be used in combination with a secondary linker is asubstituted silane. An example of such a suitable substituted silane is,without limitation, aminopropyltriethoxysilane. Examples of suitablesecondary linkers are, without limitation, homo- and/orhetero-bifunctional cross-linking agents and hydrosilylated alkenes andalkynes.

[0062] Because secondary linkers typically cannot bind directly to thePSc, the primary linker provides a direct interaction between the PScstructure and a reactive group of the secondary linker. Accordingly, theprimary and secondary linkers combined provide an indirect interactionbetween the PSc and the recognition element.

[0063] Also, using secondary linkers with primary linkers, provides evenlonger spacers compared to the primary linker alone. Consequently, amongother functions, primary and secondary linkers combined can reducepotential steric hindrance problems arising from an unusually bulkytarget analyte seeking to interact with a recognition element and/orPSc/RE. Secondary linkers can also provide a greater flexibility inselection of a functional group which is reactive with, for example, anamine, sulfhydryl, carbohydrate or carboxyl group of a recognitionelement.

[0064] Homo-bifunctional cross-linking agents have two similar reactivegroups. For example, a homo-bifunctional cross-linking agent can have afirst reactive group that can interact with an amine group of a primarylinker and a second reactive group that can interact with an amine groupof a recognition element. Examples of homo-bifunctional cross-linkingagents are, without limitation, glutaraldehyde, disuccinimidyl suberate,its sulfonated analog, and bis(sulfosuccinimidyl) suberate.

[0065] Hetero-bifunctional cross-linking agents have two differentreactive groups. For example, a hetero-bifunctional cross-linking agentcan have a first reactive group that can interact with an amine group ofa primary linker and a second reactive group that can interact with asulfhydryl group of a recognition element. Examples ofhetero-bifunctional cross-linking agents are succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and its sulfonatedanalog, 4-(4-N-maleimidophenyl) butyric acid hydrazide-HCl, and4-(p-azidosalicylamido)butylamine.

[0066] Other linkers which are reactive with hydroxyl groups of the PScand functional groups of the selected recognition elements will beapparent to those skilled in the art. It will also be understood bythose skilled in the art that other linkers, which are reactive withnon-hydroxyl groups (produced by other PSc stabilization techniques) andthe functional groups of the selected recognition elements, may beselected for bonding to a PSc structure.

[0067] PSc/RE Interaction with Target Analyte

[0068] Using a recognition element having a specific affinity for atarget analyte will substantially reduce the likelihood that non-targetcompounds in the sample mixture will interact with the recognitionelement. Also, using a recognition element that facilitates acharacteristic modulation in the PL response of the PSc/RE when thetarget analyte interacts with the PSc/RE will substantially reduce thelikelihood of “false” modulations produced by non-target compounds.Accordingly, the effect, if any, of non-target compounds in a samplemixture on the PL response can be significantly reduced.

[0069] Likewise, in a preferred embodiment where the recognition elementhas a specific affinity for the target analyte, the target analyte willpreferentially interact with the recognition element rather than withthe PSc structure.

[0070] The PSc structures of the present invention may be exposed to asample containing a target analyte in a number of differentapplications, examples of which are provided below. In someapplications, it may be desirable to suspend the PSc/RE in a suitablecarrier to allow for increased interaction of the PSc/RE with the targetanalyte. Using contacting means that will be apparent to those skilledin the art, the carrier/PSc/RE mixture can then enhance the extent ofcontact between an unknown composition or organism, whether plant oranimal, containing the target analyte as well as non-target compounds.Such a carrier may include an agent to reduce the carrier's surfacetension, increase its hydrophilicity or increase its hydrophobicity, asappropriate. Such agents include, without limitation, alcohols,detergents and organic solvents at a concentration sufficient to achievethe desired effect without adversely affecting the efficacy of thePSc/RE.

[0071] Linker Treatment

[0072] Linkers may be treated to (a) enhance the preferentialinteraction between the linker and the recognition element, and/or (b)inhibit interaction between unassociated linkers (i.e., linkers notassociated with a recognition element but still linked to the PSc) andnon-target compounds and/or target analytes.

[0073] Preferential interaction between the linker and the recognitionelement may be enhanced by providing a stronger association between therecognition element and the linker and/or by orienting the recognitionelement for better interaction with a target analyte.

[0074] Interaction between unassociated linkers and non-target compoundsand/or target analytes may be inhibited by blocking reactive groups ofunassociated linkers, and/or by attaching to the reactive groups ofunassociated linkers a moiety that has an affinity for a compound notpresent in a target analyte composition.

[0075] Non-limiting examples of such linker treatments, discussed morefully below, are (1) immunoglobulin binding proteins, (2) a biotinreactive agent, and (3) blocking solutions, e.g. amine buffer solutions.

[0076] Immunoglobulin Binding Protein Treatment

[0077] In one embodiment, linkers attached to a PSc structure may betreated with an immunoglobulin binding protein (“IgBP”), (e.g., aProtein A, Protein G or Protein L). IgBP has a specific affinity for aselected portion of an antibody known as the Fc domain. Consequently,because all antibodies have an Fc domain, an antibody recognitionelement will preferentially interact with IgBP. Therefore, when theantibody recognition element is contacted with the IgBP-treated linkerattached to the PSc structure, the Fc domain of the antibody recognitionelement interacts with the IgBP-treated linker.

[0078] Because the IgBP has a specific affinity for the Fc domain ofantibodies, IgBP treatment of the linkers reduces binding of non-targetcompounds and target analytes to the linkers, assuming such compoundsand analytes have no Fc domain, of course. Another advantage of IgBPlinker treatment is that the antibody recognition elements are properlyoriented for antigen target analyte interaction. Specifically, the Fcdomain of the antibody recognition element binds to the IgBP, while theFab domains of the antibody remain available for antigen interaction.

[0079] Biotin Reactive Agent Treatment

[0080] In another embodiment, linkers attached to a PSc structure may betreated with a biotin reactive agent (“BRA”), which has an affinity forbiotin. Non-limiting examples of biotin reactive agents arestreptavidin, neutravidin and avidin. In this case, the desiredrecognition elements are treated with biotin to produce a biotinylatedrecognition element. Accordingly, when the biotinylated recognitionelement is contacted with the BRA-treated linker attached to the PScstructure, the biotin moiety on the recognition element willpreferentially interact with the BRA.

[0081] Because the BRA has an affinity only for biotin, BRA treatment ofthe linkers reduces binding of non-target compounds and target analytes,which do not have biotin moieties, to the linkers. Another advantage ofthe BRA linker treatment is a slightly stronger association between therecognition elements and the linkers attached to the PSc structure.Furthermore, the biotinylated recognition element is still capable ofinteracting with a target analyte.

[0082] Blocking Solution Treatment

[0083] In a further embodiment, the PSc/RE may be treated with ablocking solution to specifically block reactive groups of unreactedlinkers against binding with non-target compounds and target analytes.Blocking solutions include, for example, amine buffers, such as aglycine-buffered solution. Any unreacted linker amine reactive groupsare thus blocked by the amine groups of the blocking solution. However,the blocking solution does not block the receptor or acceptor region ofthe recognition element, which remain available for target analyteinteraction.

[0084] PL Detection

[0085] An embodiment of an apparatus 10 which may be used to detect PLis illustrated schematically in FIG. 1. A sample 19 is placed on asample holder 20. Light at a predetermined wavelength is directed at thesample from an argon ion laser 12. Light passes through a narrow bandfilter 14, which reduces any noise radiation caused by gas fluorescencein the chamber of the laser 12. The filter 14 absorbs radiation of allwavelengths except for light having the predetermined wavelength. Achopper 16 is used to eliminate any constant noise caused by peripherallight radiation, if any. The chopper 16 provides time modulation of thelaser radiation with a certain frequency, which enables separation of aPL signal from the background noise at an amplifier 42. After thechopper 16, light passes through a lens 17 to the sample 19. Lightradiation scattered by the sample 19 is directed through lenses 18 and abroad band filter 22 to an input slit 32 and a mirror 34 in amonochromator 30. The broad band filter 22 absorbs radiation ofwavelengths outside a predetermined range and thus reduces laserradiation reflected from optical elements of the apparatus 10.

[0086] A rotating diffraction grating 36 located inside themonochromator 30 allows for angular separation of different wavelengthsλ₁, λ₂, λ₃ and λ₄ of a scattered beam spectrum to a mirror 35. Rotationof the grating 36 is controlled by a computer 40 to separate differentspectral components of an output slit 33 of the monochromator 30.Intensities of spectral components λ₁, λ₂, λ₃ and λ₄ are recorded by aphotodiode 38. The signals are amplified by an amplifier in thephotodiode 38 and the amplifier 42 and acquired by the computer 40. Allmeasurement processes are controlled by the computer 40.

[0087] Quantifying the concentration of a target analyte may bedetermined using a calibration curve of PL intensity for knownconcentrations of target analyte or using different concentrations of arecognition element.

[0088] Applications

[0089] The PSc/RE structures may be used alone or in combination with awide array of devices used to support a diversity of applications,including, without limitation, biotechnology, such as biosensors formedical, environmental, industrial and defense applications (e.g.chemical and biological warfare agent detection/identification),genomics, diagnostics, and other molecular/cell biological areas, suchas cell isolation/sorting, microstructural cell analysis, etc.

[0090] For example, there is an increasing demand for high throughputscreening devices for genomics and diagnostic screening. In theinvention, separate batches of PSc/RE structures having differentrecognition elements can be prepared and then incorporated and/orblended in predetermined ratios into “biochips” or microcapillary arrayconfigurations, discussed more fully below. Samples can be contactedwith immobilized or suspended PSc/RE structures in such configurationsand interaction with a target analyte can be readily determined asdiscussed above. Advantageously, 3-D PSc structures can be used withconventional hardware, for example automated microvolume dispensers, toform analyte detection and/or identification micro-arrays.

[0091] In one embodiment, PSc/RE structures are advantageously used on aphoton detection “biochip” where a variety of recognition elements canbe placed in different regions of the same device. To date, thepractical issue of having multiple but different recognition elements ona small semiconductor chip has not yet been satisfactorily met due to 1)insufficient signal intensity for detection of target analyteinteraction, and 2) the difficulty of having multiple but differentrecognition elements deposited within a small area. Small 3-D PScstructures, described above, are particularly suitable for this type ofapplication. A sample containing an unknown compound, but which issuspected of being one of a number of compounds, can thus be identifiedby contacting a biochip having recognition elements for the suspectedcompounds. When a region of the biochip produces a modulated PLresponse, the compound is identified by the recognition element used inthat particular area.

[0092] PSc/RE structures can also be arranged in micro-arrays, eachseparate area containing a specified amount of a PSc/RE structure, andeach separate area having a specificity for a different target analyte,or the same target analyte, such that binding of the target analyte(s)results in PL modulation, for example in a unique pattern (patternrecognition).

[0093] In another embodiment, PSc/RE structures can be used as a packingin a micro-capillary column which can allow for the flow-through ofcarrier liquids containing one or more target analytes. Themicro-capillary columns could be optically clear to detect PL modulationupon interaction with a target analyte. Parallel or sequential placementof micro-capillary columns could be used to detect and identify one ormore target analytes. In another configuration, the micro-capillarycolumns could also incorporate fiber optic elements allowing fordetection of such PL modulation.

[0094] In another embodiment, the target analyte may be found in or onan organism or portion of an organism. By “organism”, we mean microbialcells, animal and plant cells and tissues, microbial spores, viruses andthe like. For example, without limitation, the target analyte may abacteria, a virus or a microbial spore. Examples of such target analytesinclude anthrax spores and E. coli, such as E. coli 0157 (a causativeagent of hamburger disease). PSc/RE structures, which have an affinityfor one of such target analytes are contacted with a sample suspected ofhaving such a target analyte or a sample which is being tested for thepresence of such a target analyte. In the presence of such a targetanalyte, one or more recognition elements attach itself to the cell wallor coating through exterior receptors of the cells. Advantageously,these PSc structures have a diameter or largest dimension of about 100nm to about 1 micron.

[0095] It is possible that a PSc/RE could be injected or otherwiseinserted in an organism for detection of an internal or intracellulartarget analyte. It is also possible that an organism could be induced toingest or that an organism actively ingests, for example byphagocytosis, a PSc/RE for detection of an internal or intracellulartarget analyte.

[0096] The following non-limiting examples of embodiments of the presentinvention that may be made and used as claimed herein are provided forillustrative purposes only.

EXAMPLES Example 1 Production of PSi Particles

[0097] Small particles of silicon were produced by mechanicalfragmentation of planar n-type silicon obtained from Silicon Quest(Santa Clara, Calif., USA). Particles were produced by mechanicalfragmentation using a mortar and pestle to produce granular particles ofan irregular shape. This procedure produced particles with a wide rangeof sizes. Particles with a diameter, or other largest dimension, between30 and 1000 μm were selected by mechanical sieving through a polymermesh membrane. The particles were analyzed by BET(Brunnauer-Emmett-Toller) analysis.

[0098] BET analysis was made using a MICROMERITICS ASAP 2000™ instrument(Norcross, Ga., USA). Empty sample tubes were weighed and then reweighedafter transfer of samples to the sample tubes. The samples were placedon the instrument and degassed by heating to 180° C. and placing undervacuum overnight. After degassing, the samples were removed from theinstrument and re-weighed. The samples were then analyzed and re-weighedafter analysis. This last recorded weight was used in analysiscalculations. Volume absorbed points were taken from 0.05 to 0.3relative pressure.

[0099] The BET surface area of the particles, based on a 5-point BETsurface area analysis, was determined to be 0.1724±0.0016 m²/g with acorrelation coefficient of 0.999871.

[0100] The silicon particles were made porous, with a random poredistribution, using a chemical etching method. The silicon particleswere suspended in an acidic solution of 70% nitric acid, 50%hydrofluoric acid and water in a 1:4:1 ratio for 60 seconds at roomtemperature. The reaction produced hydrogen gas and caused violentmixing of the solution, so that no additional mixing was required tokeep the particles in suspension. The etching reaction was stopped bydilution of the acidic solution with water.

[0101] The resultant PSi particles were analyzed using Scanning ElectronMicroscopy (SEM), BET and BJH (Barett-Joyner-Halenda) analysis.

[0102]FIGS. 2 and 3 are SEM micrographs of porous silicon (PSi)particles produced by the method of this example.

[0103] BET and BJH analyses were made using a MICROMERITICS ASAP 2000™instrument (Norcross, Ga., USA) using the procedure described above.Adsorption and desorption points were taken from 0.01 to 0.99 relativepressure.

[0104] The BET surface area of the particles, based on a 5-point BETsurface area analysis, was determined to be 1.8559±0.0124 m²/g with acorrelation coefficient of 0.999926. Accordingly, the surface area ofthe particles was increased by approximately 11 times using theabove-described etching technique. Based on a total pore volumedetermined by BET divided by pore area determined by BET, the averagepore diameter was 4.77075 nm.

[0105] Results of the BJH adsorption pore distribution analysis arelisted below in Table 1. TABLE 1 Pore Diameter Incremental CumulativeIncremental Cumulative Range Average Pore Volume Pore Volume Pore AreaPore Area (nm) Diameter (nm) (cm³/g) (cm³/g) (m²/g) (m²/g) 210.62-295.20238.98 0.001559 0.001559 0.026 0.026 103.15-210.62 123.50 0.0000710.001631 0.002 0.028  81.15-103.15 89.50 0.000027 0.001657 0.001 0.03040.42-81.15 48.22 0.000077 0.001734 0.006 0.036 27.18-40.42 31.200.000049 0.001784 0.006 0.042 20.35-27.18 22.76 0.000031 0.001814 0.0050.048 16.32-20.35 17.87 0.000041 0.001855 0.009 0.057 13.58-16.32 14.690.000026 0.001881 0.007 0.064 11.31-13.58 12.23 0.000036 0.001917 0.0120.076 10.55-11.31 10.90 0.000013 0.001930 0.005 0.080  8.09-10.55 8.970.000079 0.002009 0.035 0.116 6.65-8.09 7.22 0.000067 0.002076 0.0370.153 5.64-6.65 6.05 0.000156 0.002233 0.103 0.256 4.85-5.64 5.180.000165 0.002397 0.127 0.383 4.23-4.85 4.49 0.000224 0.002622 0.2000.583 3.72-4.23 3.94 0.000229 0.002851 0.232 0.816 3.29-3.72 3.480.000221 0.003072 0.255 1.070 2.93-3.29 3.08 0.000193 0.003264 0.2501.320 2.61-2.93 2.74 0.000158 0.003422 0.230 1.550 2.32-2.61 2.440.000128 0.003550 0.210 1.760 2.06-2.32 2.17 0.000116 0.003666 0.2141.974 1.82-2.06 1.92 0.000095 0.003762 0.198 2.172 1.72-1.82 1.770.000040 0.003802 0.092 2.264

[0106] Results of the BJH desorption pore distribution analysis arelisted below in Table 2. TABLE 2 Pore Diameter Incremental CumulativeIncremental Cumulative Range Average Pore Volume Pore Volume Pore AreaPore Area (nm) Diameter (nm) (cm³/g) (cm³/g) (m²/g) (m²/g) 278.56-294.91286.27 0.001155 0.001155 0.016 0.016  87.25-278.56 103.77 0.0005040.001659 0.019 0.036 62.76-87.25 71.02 0.000040 0.001700 0.002 0.03835.37-62.76 41.80 0.000065 0.001765 0.006 0.044 24.52-35.37 27.970.000050 0.001815 0.007 0.051 20.20-24.52 21.93 0.000033 0.001849 0.0060.057 16.09-20.20 17.66 0.000038 0.001887 0.009 0.066 12.69-16.09 13.980.000041 0.001928 0.012 0.078 10.89-12.69 11.65 0.000026 0.001954 0.0090.087  9.91-10.89 10.35 0.000024 0.001978 0.009 0.096 7.81-9.91 8.600.000059 0.002037 0.027 0.123 6.40-7.81 6.95 0.000075 0.002112 0.0430.166 5.36-6.40 5.78 0.000096 0.002208 0.067 0.233 4.57-5.36 4.900.000124 0.002332 0.101 0.334 3.95-4.57 4.21 0.000184 0.002515 0.1740.508 3.44-3.95 3.66 0.000222 0.002738 0.243 0.752 3.01-3.44 3.200.000270 0.003008 0.337 1.089 2.65-3.01 2.81 0.000262 0.003270 0.3731.462 2.29-2.65 2.44 0.000172 0.003442 0.282 1.744 2.00-2.29 2.120.000120 0.003562 0.226 1.970 1.74-2.00 1.85 0.000101 0.003663 0.2192.189

[0107] The above results illustrate that there are no inkwell or otherretaining pores in the sample analyzed. The BJH cumulative adsorptionpore volume of pores having a pore diameter between 1.7 and 300 nm was0.003802 cm³/g, while the BJH cumulative desorption pore volume was0.003663 cm³/g. The BJH cumulative adsorption surface area of poreshaving a pore diameter between 1.7 and 300 nm was 2.2636 m²/g, while theBJH cumulative desorption surface area was 2.1889 m²/g.

[0108] Based on the total pore volume as determined by BJH divided bypore volume as determined by BJH, the average adsorption and desorptionpore diameters were determined to be 6.71859 nm and 6.69407 nm,respectively.

[0109] The differences in surface area and pore diameter values betweenBET and BJH analyses appear to be due to the calculation method used forBJH analysis, which assumes that the pores are a bundle of cylinders.However, the SEM micrographs produced show that the porous texture ofthe particles cannot properly be characterized as a bundle of cylinders.Accordingly, the BET surface area analysis is probably a more accuratedetermination of the surface area.

Example 2 Production of PSi Particles

[0110] PSi particles were prepared as in Example 1, except that thesilicon particles were suspended in the acidic solution for 30 seconds.The resultant PSi particles were analyzed by BET analysis.

[0111] BET analysis was made using a MICROMERITICS ASAP 2000™ instrument(Norcross, Ga., USA), using the procedure described above. Volumeabsorbed points were taken from 0.05 to 0.3 relative pressure.

[0112] The BET surface area of the particles, based on a 5-point BETsurface area analysis, was determined to be 0.6858±0.0039 m²/g with acorrelation coefficient of 0.999934. Accordingly, the surface area ofthe particles was increased by approximately 4 times using theabove-described etching technique.

[0113]FIGS. 4 and 5 are SEM micrographs of PSi particles produced by themethod of this example.

Example 3 Oxidation of PSi Particles Using Peroxide

[0114] PSi particles produced in Example 1 were subjected to chemicaloxidation using peroxide. The PSi particles were incubated at roomtemperature for 1 hour in an aqueous solution of 30% peroxide.

Example 4 Oxidation of PSi Particles Using DMSO

[0115] PSi particles produced in Example 1 were subjected to chemicaloxidation using dimethylsulfoxide (DMSO) alone and a solution of DMSOcontaining 500 mg/ml of 2,6-di-tert-butyl4-methylphenol (BHT), a freeradical scavenger. The PSi particles were incubated at room temperaturefor 1 hour in DMSO and 2 hours in the solution of DMSO/BHT.

Example 5 Oxidation of PSi Particles Using Iodine Chips

[0116] PSi particles produced in Example 1 were subjected to chemicaloxidation using iodine chips. The PSi particles were incubated in thepresence of iodine chips either (1) in vacuum or (2) in air.

[0117] In vacuum, the particles were incubated under vacuum in a vacuumflask containing the iodine chips for 2 hours at room temperature. Theparticles were then exposed overnight to air at room temperature.

[0118] In air, the particles were incubated in a stoppered flaskcontaining iodine chips for 2 hours at room temperature. The particleswere then exposed overnight to air at room temperature.

Example 6 Attachment of Primary Linker

[0119] Oxidized particles prepared in Example 3 were immersed in a 10%(v/v) solution of 3-glycidoxypropyltrimethoxysilane, a primary linker,in water for 4 hours at 75° C., followed by annealing at 110° C.overnight to form a covalent bond between the3-glycidoxypropyltrimethoxysilane and the hydroxyl groups on the PSisurface.

Example 7 Attachment of Primary and Secondary Sulfo-SMCC Linkers

[0120] Oxidized particles prepared in Example 3 were immersed in a 10%(v/v) solution of aminopropyltriethoxysilane, a primary linker, in waterfor 4 hours at 75° C., followed by annealing at 110° C. overnight toform a covalent bond between the aminopropyltriethoxysilane and thehydroxyl groups on the PSi surface.

[0121] The particles were then immersed in a solution ofsulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(sulfo-SMCC), a heterobifunctional secondary linker, containing 10 mgsulfo-SMCC per ml of water, for 1 hour at room temperature to formcovalent bonds between the primary linker and the secondary linker.After incubation with the sulfo-SMCC solution, the particles were washedwith water.

Example 8 Attachment of Primary and Secondary Glutaraldehyde Linkers

[0122] Oxidized particles prepared in Example 3 were immersed in a 10%(v/v) solution of aminopropyltriethoxysilane, a primary linker, in waterfor 4 hours at 75° C., followed by annealing at 110° C. overnight toform a covalent bond between the aminopropyltriethoxysilane and thehydroxyl groups on the PSi surface.

[0123] The particles were then immersed in a solution of glutaraldehyde(2.5% in phosphate buffer), a homobifunctional secondary linker, for 1hour at room temperature to form covalent bonds between the primarylinker and the secondary linker. After incubation with theglutaraldehyde solution, the particles were washed with water.

Example 9 Attachment of Primary and Secondary BS³ Linkers

[0124] Oxidized particles prepared in Example 3 were immersed in a 10%(v/v) solution of aminopropyltriethoxysilane, a primary linker, in waterfor 4 hours at 75° C., followed by annealing at 110° C. overnight toform a covalent bond between the aminopropyltriethoxysilane and thehydroxyl groups on the PSi surface.

[0125] The particles were then immersed in a solution ofbis(sulfosuccinimidyl)suberate (BS³), a homobifunctional secondarylinker, at a concentration of 5 mg per ml of water, for 1 hour at roomtemperature to form covalent bonds between the primary linker and thesecondary linker. After incubation with the BS³ solution, the particleswere washed with water.

Example 10 Attachment of Antibody Recognition Element

[0126] Purified mouse immunoglobulin G (IgG) obtained from Sigma-AldrichCanada Ltd. (Oakville, Ontario, Canada) was attached to the primarylinker (3-glycidoxypropyltrimethoxysilane) of the particles produced inExample 6 by incubation of the IgG, in a phosphate buffered salinesolution, at 37° C. for 90 minutes.

[0127] For visualization (microscopy) and quantitative determination(fluorometry) of antibody attachment, a specific anti-mouse IgG F(ab′)₂fragment, conjugated with Cy3 fluorescent marker (red fluorescence,obtained from Sigma-Aldrich Canada Ltd.), was contacted with theparticles having the IgG antibody attached. The fluorescently-labeledanti-mouse IgG F(ab′)₂ fragment, in a phosphate buffered salinesolution, was incubated in the presence of the particles having the IgGattached for 30 minutes at room temperature. The formation, by affinitybinding, of the complex mouse IgG/anti-mouse IgG F(ab′)₂ fragment wasexamined by epi-fluorescence microscopy and quantified by fluorometry.FIG. 6 is an epi-fluorescence micrograph (Nikon Diaphot 300 microscope)at 200× magnification, showing the interaction of antigen to PSiparticles modified with IgG antibody.

Example 11 Attachment of Antibody Recognition Element

[0128] Purified mouse immunoglobulin G (IgG) obtained from Sigma-AldrichCanada Ltd. (Oakville, Ontario, Canada) was partially reduced with Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), at room temperature for25 minutes, to expose the immunoglobulin sulfhydryl groups. The reducedIgG was attached, via the sulfhydryl reactive groups, to the secondarylinker (sulfo-SMCC) of the particles produced in Example 7 by incubationof the reduced IgG, in a phosphate buffered saline solution, at 37° C.for 90 minutes.

[0129] For visualization (microscopy) and quantitative determination(fluorometry) of antibody attachment, a specific anti-mouse IgG F(ab′)₂fragment, conjugated with Cy3 fluorescent marker, was contacted with theparticles having the IgG antibody attached. The fluorescently-labeledanti-mouse IgG F(ab′)₂ fragment, in a phosphate buffered salinesolution, was incubated in the presence of the particles having thereduced IgG attached for 30 minutes at room temperature. The formation,by affinity binding, of the complex mouse IgG/anti-mouse IgG F(ab′)₂fragment was examined by epi-fluorescence microscopy and quantified byfluorometry, which showed good coverage and distribution of antibodyrecognition element on PSi particles.

Example 12 Attachment of Antibody Recognition Element

[0130] Purified mouse immunoglobulin G (IgG) obtained from Sigma-AldrichCanada Ltd. was attached to the secondary linker (glutaraldehyde) of theparticles produced in Example 8 by incubation of the reduced IgG, in aphosphate buffered saline solution, at 37° C. for 90 minutes.

[0131] For visualization (microscopy) and quantitative determination(fluorometry) of antibody attachment, a specific anti-mouse IgG F(ab′)₂fragment, conjugated with Cy3 fluorescent marker, was contacted with theparticles having the IgG antibody attached. The fluorescently-labeledanti-mouse IgG F(ab′)₂ fragment, in a phosphate buffered salinesolution, was incubated in the presence of the particles having the IgGattached for 30 minutes at room temperature. The formation, by affinitybinding, of the complex mouse IgG/anti-mouse IgG F(ab′)₂ fragment wasexamined by epi-fluorescence microscopy and quantified by fluorometry.

[0132] Comparable results were obtained for oxidized particles producedin Examples 4 and 5 and treated with the primary and secondary linkersin Example 8.

Example 13 Attachment of Enzyme Recognition Element

[0133] Acetylcholinesterase enzyme (Sigma-Aldrich Canada Ltd.) wasattached through the glutaraldehyde secondary linker of the particlesproduced in Example 8 by incubation of the enzyme, in a phosphatebuffered saline solution, with the particles at room temperature for 90minutes.

[0134]FIG. 7 graphically compares acetylcholine hydrolysis for a controlenzyme versus enzyme bound to the enzyme-modified PSi.

Example 14 Treatment with Protein A, Attachment of Mouse IgG Antibody

[0135] Particles produced in Example 8 were treated with Protein A byincubating the particles in a solution of Protein A in a phosphatebuffered saline solution for 3 hours at 37° C. After incubation, theparticles were washed with a phosphate buffered solution.

[0136] Purified mouse IgG antibody (obtained from Sigma-Aldrich CanadaLtd.) was attached to the Protein A treated particles by incubating theparticles in a phosphate buffered saline solution of IgG for 90 minutesat 37° C.

[0137] For visualization (microscopy) and quantitative determination(fluorometry) of IgG attachment, a specific anti-mouse IgG F(ab′)₂fragment, conjugated with Cy3 fluorescent marker, was contacted with theparticles having the IgG antibody attached. The fluorescently-labeledanti-mouse IgG F(ab′)₂ fragment, in a phosphate buffered salinesolution, was incubated in the presence of the particles having the IgGattached for 30 minutes at room temperature. The formation, by affinitybinding, of the complex IgG/anti-mouse IgG F(ab′)₂ fragment, used asantigen, was examined by epi-fluorescence microscopy and quantified byfluorometry.

[0138] Both epi-fluorescence microscopy and fluorometry showed goodcoverage and distribution of antibody recognition elements on PSiparticles.

[0139] The analytes conjugated with the fluorescent markers were alsoused to determine whether the Protein A-treatment minimized non-specificbinding of biomolecules to the Protein A-treated PSi surface, other thanthrough the immunoglobulin attached above. Both epi-fluorescencemicroscopy and fluorometry showed no significant non-specific binding ofbiomolecules other than immunoglobulins with intact Fc domain.

Example 15 Treatment with Protein A, Attachment of Mouse Collagen IV

[0140] Particles produced in Example 8 were treated with Protein A byincubating the particles in a solution of Protein A in a phosphatebuffered saline solution for 3 hours at 37° C. After incubation, theparticles were washed with a phosphate buffered solution.

[0141] Purified mouse collagen IV antibody (obtained from Sigma-AldrichCanada Ltd.) was attached to the Protein A treated particles byincubating the particles in a phosphate buffered saline solution of themouse collagen IV antibody for 90 minutes at 37° C.

[0142] For visualization (microscopy) and quantitative determination(fluorometry) of mouse collagen IV antibody attachment, a specificanti-mouse IgG F(ab′)₂ fragment, conjugated with fluoresceinisothiocyanate (FITC, green fluorescence) fluorescent marker, obtainedfrom Sigma-Aldrich Canada Ltd., was contacted with the particles havingthe antibody attached. The fluorescently-labeled anti-mouse IgG F(ab′)₂fragment, in a phosphate buffered saline solution, was incubated in thepresence of the particles having the antibody attached for 30 minutes atroom temperature. The formation, by affinity binding, of the mousecollagen IV antibody/anti-mouse IgG F(ab′)₂ fragment complex wasexamined by epi-fluorescence microscopy and quantified by fluorometry.

[0143] Both epi-fluorescence microscopy and fluorometry showed goodcoverage and distribution of antibody recognition elements on PSiparticles.

[0144] The analytes conjugated with the fluorescent markers were alsoused to determine whether the Protein A-treatment minimized non-specificbinding of biomolecules to the Protein A-treated PSi surface, other thanthrough the immunoglobulin attached above. Both epi-fluorescencemicroscopy and fluorometry showed no significant non-specific binding ofbiomolecules other than immunoglobulins with intact Fc domain.

Example 16 Treatment with Blocking Solution

[0145] Minimization of non-specific binding by using a blocking solutionwas tested on the particles produced in Examples 6, 8 and 9. Theparticles were treated with a glycine buffer (either 50 mM or 200 mM ateither pH 8.6 or pH 10) for 30 and 60 minutes at room temperature. Afterincubation of the particles in glycine buffer, mouse IgG (used here as atest substance having amine reactive groups) conjugated with Cy3fluorescent marker was incubated with the particles at 37° C. for 90minutes. After protein incubation, the particles were washed with aphosphate buffered saline solution. Determination of the blocking effectof the glycine buffer, i.e. the ability of the buffer to inhibit bindingof protein on the linkers, was done by assessment of thefluorescently-labeled IgG binding on the particles. Bothepi-fluorescence microscopy and fluorometry showed a significantlyreduced, >70%, binding of antibodies on the treated PSi particles.Similar results were obtained for particles produced in Examples 6, 8and 9.

[0146]FIG. 8 is an epi-fluorescence micrograph at 200× magnificationwhich shows binding of antibody to PSi not treated with glycine buffer.FIG. 9 is an epi-fluorescence micrograph at 200× magnification whichdemonstrates the reduction in binding of antibody of PSi treated withglycine buffer.

[0147] In practice, recognition elements would be attached to the PScparticles prior to treatment with the blocking solution. The blockingsolution would not interfere with binding of a target analyte to therecognition element, but would minimize binding of non-target compoundsand the target analyte directly to the surface of the PSc.

[0148] In order to verify that the glycine buffer has no detrimentaleffect on the ability for IgG to interact with an antigen, a specificanti-mouse IgG F(ab′)₂ fragment, conjugated with fluoresceinisothiocyanate fluorescent marker was added to the IgG for a 30 minuteincubation at room temperature. The binding activity of IgG, followingincubation in glycine buffer, was assessed by epi-fluorescencemicroscopy and fluorometry. No detrimental effect of the glycine bufferon the subsequent affinity binding activity of IgG was observed.

[0149]FIG. 10 is an epi-fluorescent micrograph at 200× magnificationwhich shows binding activity of antibody in FIG. 9 is not adverselyaffected by glycine buffer.

Example 17 Characterization of Photoluminescence

[0150] The apparatus depicted in FIG. 1 was used to analyzephotoluminescence of a number of samples produced in the above Examples.Light having a wavelength of 488 nm was directed at the sample. Thespectral sensitivity of the photodiode was in the range of from about420 nm to about 1100 nm.

[0151] The photoluminescence of PSi particles produced in Example 1 wasanalyzed and the PL intensity is graphically represented as a functionof photon energy and wavelength in FIG. 11. The PL intensity maximum wasabout 625 arbitrary units at about 1.94 eV.

[0152] The photoluminescence of PSi particles having an antibodyrecognition element attached thereto, produced in Example 8, was thenanalyzed. The PL intensity is graphically represented as a function ofphoton energy and wavelength in FIG. 12. The PL intensity maximum wasabout 1000 arbitrary units at about 2.24 eV.

[0153] The photoluminescence of PSi particles having an antibodyrecognition element attached thereto, produced in Example 8, and ananti-mouse IgG target analyte attached to the recognition element wasthen analyzed. The PL intensity is graphically represented as a functionof photon energy and wavelength in FIG. 13. The PL intensity maximum wasabout 1900 arbitrary units at about 2.27 eV.

[0154] The photoluminescence patterns discussed above show significant,distinct and reproducible PL pattern modulations. The expectedluminescent signal, of lower energy in the orange-red region of thevisible spectrum, emitted by the PSi particle of Example 1 show anincrease in energy, in the yellow region of the spectrum, on attachmentof a recognition element and a distinct higher energy signal in thegreen region of the spectrum generated by attachment of the targetanalyte to the recognition element. The photoluminescence emission,measured using the apparatus of FIG. 1 described above, is intense andreadily distinguishable by eye, i.e. there was strong visible lightemission.

[0155] Preferred embodiments of the present invention have beendescribed. It will be understood that the foregoing is illustrative onlyand that other embodiments and applications can be employed withoutdeparting from the true scope of the invention described in thefollowing claims.

1-30. Cancel
 31. A method for detecting a target analyte comprising: (a)irradiating a complex with at least one wavelength of electromagneticradiation in the range of from about 100 nm to about 1000 nm, whereinsaid complex comprises a sample suspected of containing said targetanalyte and a sensor composition, and (b) detecting a luminescentresponse in the range of from about 200 nm to about 800 n, wherein saidsensor composition comprises at least one porous semiconductor materialmodified with at least one recognition element, wherein said recognitionelement is capable of specifically binding to said target analyte, andwherein said luminescent response is produced by said poroussemiconductor composition and is modified upon binding of said targetanalyte to said recognition element.
 32. The method according to claim31, wherein the intensity of said luminescent response is modified uponbinding of said target analyte to said recognition element.
 33. Themethod according to claim 31, wherein the wavelength of said luminescentresponse is modified upon binding of said target analyte to saidrecognition element.
 34. The method according to claim 31, wherein saidrecognition element is selected from the group consisting of proteins,nucleic acids, oligonucleotides, lectins, carbohydrates, glycoproteins,lipids and combinations thereof.
 35. The method according to claim 34,wherein said recognition element is a protein.
 36. The method accordingto claim 35, wherein said recognition element is an antibody or antibodyfragment.
 37. The method according to claim 31, wherein said targetanalyte is selected from the group consisting of inorganic, organic andbiomolecular compounds.
 38. The method according to claim 37, whereinsaid target analyte is selected from the group consisting of proteins,polypeptides, peptides, toxins, metabolic regulators, enzyme substrates,complementary oligonucleotide sequences; single-stranded DNA,single-stranded RNA; hormonal receptor ligands and lectin ligands. 39.The method according to claim 38, wherein said target analyte isselected from the group consisting of antigens, nerve agents,pesticides, and insecticides;.
 40. The method according to claim 31,wherein said recognition element is covalently bonded to saidsemiconductor material.
 41. The method according to claim 40, whereinsaid recognition element is bound to said semiconductor material via atleast one primary linker.
 42. The method according to claim 31, whereinsaid semiconductor material is selected from the group consisting ofsilicon, silicon carbide, silicon dioxide, germanium, gallium, galliumarsenide, silicon gallium phosphide, cadmium, selenium, copper oxide andcombinations thereof.
 43. The method according to claim 42, wherein saidsemiconductor material further comprises a dopant for said semiconductormaterial.
 44. A method for detecting an organism comprising: (a)irradiating a complex with at least one wavelength of electromagneticradiation in the range of from about 100 nm to about 1000 nm, whereinsaid complex comprises a sample suspected of containing said organismand a sensor composition, and (b) detecting a luminescent response inthe range of from about 200 nm to about 800 nm, wherein said sensorcomposition comprises at least one porous semiconductor materialmodified with at least one recognition element, wherein said recognitionelement is capable of specifically binding to said organism, and whereinsaid luminescent response is produced by said porous semiconductorcomposition and is modified upon binding of said organism to saidrecognition element.
 45. The method according to claim 44, wherein theintensity of said luminescent response is modified upon binding of saidtarget analyte to said recognition element.
 46. The method according toclaim 44, wherein the wavelength of said luminescent response ismodified upon binding of said target analyte to said recognitionelement.
 47. The method according to claim 44, wherein said recognitionelement is selected from the group consisting of proteins, lectins,carbohydrates, glycoproteins, and combinations thereof.
 48. The methodaccording to claim 45, wherein said recognition element is a protein.49. The method according to claim 48, wherein said recognition elementis an antibody or antibody fragment.
 50. The method according to claim44, wherein said target analyte is an organism selected from the groupconsisting of bacteria, viruses, yeast, fingi and microbial spores.